Micro-organisms controlling plant pathogens

ABSTRACT

The invention relates to control of pathogen caused diseases on leaves, fruits and ears in plants, such as apple scab ( Venturia inaequalis  by treatment of plant with an isolate of  Cladosporium cladosporioides . The treatment is effective in both prevention and treatment of the fungal infection.

RELATED APPLICATIONS

This application is the United States National Stage of InternationalApplication No. PCT/NL20081050794, filed Dec. 12, 2008, which waspublished as International Publication No. WO 2009/078710, and whichclaims benefit of European Patent Application No. 07123275.5 filed Dec.14, 2007. Both applications are incorporated in reference in theirentirety herewith.

FIELD OF THE INVENTION

The invention relates to the field of plant pathogen control,particularly on leaves, fruits or ears, more particularly in fruitcrops, such as apple, and particularly to the control of the fungalfoliar disease apple scab (Venturia inaequalis).

BACKGROUND OF THE INVENTION

Apple scab is of major economic importance in the areas where apples aregrown. If not controlled, the disease can cause extensive losses (70percent or greater) where humid, cool weather occurs during the springmonths. Losses result directly from fruit or pedicel infections, orindirectly from repeated defoliation which can reduce tree growth andyield.

Apple scab (see FIG. 1) can be observed on leaves, petioles, blossoms,sepals, fruit, pedicels, and less frequently, on young shoots and budscales. The first lesions are often found on the lower surfaces ofleaves as they emerge and are exposed to infection in the spring. Later,as the leaves unfold, both surfaces are exposed and can become infected.Young lesions are velvety brown to olive green and have feathery,indistinct margins. With time, the margins become distinct, but they maybe obscured if several lesions coalesce. As an infected leaf ages, thetissues adjacent to the lesion thicken, and the leaf surface becomesdeformed. Young leaves may become curled, dwarfed, and distorted wheninfections are numerous. The lesions may remain on the upper and lowerleaf surface for the entire growing season; occasionally, the underlyingcells turn brown and die, so that brown lesions are visible on bothsurfaces. The number of lesions per leaf may range from one or two tomore than a hundred. The term “sheet scab” is often used to refer toleaves with their entire surfaces covered with scab. Young leaves withsheet scab often shrivel and fall from the tree. Infections of petiolesand pedicels result in premature abscission of leaves and fruit,respectively. In late summer or early fall, lesions may appear whitishdue to the growth of a secondary fungus on the lesion surface.

Lesions on young fruit appear similar to those on leaves, but as theinfected fruit enlarge, the lesions become brown and corky. Infectionsearly in the season can cause fruit to develop unevenly as uninfectedportions continue to grow. Cracks then appear in the skin and flesh, orthe fruit may become deformed. The entire fruit surface is susceptibleto infection, but infections early in the season are generally clusteredaround the calyx end. Fruit infections that occur in late summer orearly fall may not be visible until the fruit are in storage. Thissymptom is called “pin-point” scab, with rough circular black lesionsranging from 0.004 to 0.16 inch (0.1-4 mm) in diameter.

Although research in New York has shown that the scab fungus canoverwinter in trees as conidia on bud scales, the pathogen generallyoverwinters in leaves and fruit on the orchard floor. Ascospores are themajor source of primary inoculum and are produced within pseudotheciathat develop in leaves during the winter months. In a typical year inmost locations, the first mature ascospores are capable of causinginfections at about the time of bud break or soon thereafter. Ascosporescontinue to mature and are discharged over a period of five to nineweeks, with peak discharge during the pink to petal fall phenologicalstages. The length of time required for infection to occur depends onthe number of hours of continuous wetness on the leaves and thetemperature during the wet period. Young leaves remain susceptible forfive to eight days, but their lower surfaces may become infected in latesummer. For fruit, the duration of the wet period required for infectionincreases with the age of the fruit, which remains susceptible untilharvest. Once the fungus is established in the leaf or fruit, conidiaform on the surface of the lesion and become the source of secondaryinoculum for the remainder of the season. Conidia are disseminated todeveloping leaves and fruit by splashing rain and wind. Severalsecondary cycles of conidial infection may occur during the growingseason depending upon the frequency of infection periods and thesusceptibility of host tissue.

Management of apple scab is multifaceted, with resistant cultivars,sanitation, and chemicals all being used to some degree depending on theorchard system being used and the goals of the grower.

Most of the major apple cultivars are susceptible to the fungus,although this varies somewhat. More than 25 scab-resistant cultivarshave been released, included Prima, Priscilla, Jonafree, Redfree,Liberty, Freedom, Goldrush, and Pristine. Most are adapted to the morenorthern apple-growing areas of the U.S. All scab-resistant cultivarsvary in their susceptibility to other early-season diseases; and all aresusceptible to the summer diseases. Some recently released applecultivars that have not been bred specifically for resistance to scabshow varying levels of scab susceptibility, also.

Prevention of pseudothecial formation in overwintering apple leaveswould probably eliminate scab as a serious threat to apple production.Unfortunately, complete elimination of pseudothecia is not possibleunder orchard conditions with current methods.

Apple scab is controlled primarily with fungicide sprays. A variety offungicide sprays with differing modes of action are available. When andhow they are used depends upon their mode of action. Protectantfungicides prevent the spores from germinating or penetrating leaftissue. To be effective, they must be applied to the surface ofsusceptible tissue before infection occurs. Occurrence of infection can,amongst others, be predicted with an accurate weather forecast.Protectant fungicides are applied routinely at 7 to 10 day intervals oraccording to anticipated infection periods.

Post infection fungicides control the scab fungus inside leaves andfruit. These chemicals can penetrate plant tissues to eliminate orinhibit lesion development. The ability of these fungicides to stopinfections is limited to a few hours, or up to few days (depending uponthe specific fungicide), and their effect often varies according totemperatures during the first 24 to 48 hours after infection. Somefungicides can inhibit the fungus even later into the incubation period(the time between infection and the appearance of symptoms). Eradicationof scab lesions after they appear does not usually occur, but can beachieved with the proper rate and timing of certain fungicides. Theselection of fungicides for management of scab is based on severalfactors, including the entire spectrum of other diseases that must bemanaged at that time, the potential for resistance in the scab fungus tothe selected chemical, the history of the disease in a particularorchard, the final market for the fruit, and other social and economicfactors. Good horticultural practices, such as proper site selection,tree spacing and annual pruning, facilitates better chemical control byimproving spray coverage and reducing the length of wet periods.Chemical fungicides used in the treatment or prevention of apple scabinclude maneb, mancozeb, captan, pyrimethanil and tolylfluanide.

There is thus still need for a natural fungicide, which is effective forthe control of apple scab, and which is environment-friendly andnon-toxic for humans and/or animals.

SUMMARY OF THE INVENTION

The inventors have found two new isolates of Cladosporiumcladosporioides, i.e. C. cladosporioides H39, as deposited on Dec. 13,2007 under number CBS 122244 with the Centraal Bureau Schimmelcultures,Baarn, The Netherlands and C. cladosporioides R406, as deposited on Dec.13, 2007 under number CBS 122243 with the Centraal BureauSchimmelcultures, Baarn, The Netherlands. Said isolates are useful in acomposition to control a plant pathogen of leaves, fruits and ears. Sucha composition is preferably useful for diseases wherein said plantpathogen is selected from the group consisting of apple scab (Venturiainaequalis), pear scab (Venturia pirina), leaf spot (Blumeriella jaapi),rose black spot (Diplocarpon rosae/Marssonina rosae), brown spot(Stemphylium vesicarium), powdery mildew (Podosphaeraleucotricha/Sphaerotheca pannosa), begonia mildew (Microsphaerabegoniae), strawberry powdery mildew (Sphaerotheca macularis), sootyblotch (Gloeodes pomigena), fly speck (Zygophalia jamaicensis), peachleaf curl (Taphrina deformans), brown rot/spur canker (Moniliafructigena, M. laxa), pear rust (Gymnosporangium sabinae/G. fuscum),canker (Nectria galligena), rose black spot (Diplocarpon rosae), rust onroses (Phragmidium tuberculatum/Phragmidium spp.), Botrytis spp. invarious plants, Mycosphaerella brassicicola in cabbage, Mycosphaerellafijiensis in banana, Alternaria spp. in Brassica, potato and variousother plants, Fusarium spp. especially in cereals including maize,Phytophthora infestans of potato and Plasinopara viticola in grapevine,more preferably wherein said fungal foliar pathogen is apple scab.

Further, in such a composition the Cladosporium is present as spores andfurther comprises a carrier for the Cladosporium spores, preferablywherein said carrier is glucose. Alternatively, the composition cancomprise an extract of the Cladosporium of the invention.

In another embodiment, the invention comprises a method to control apathogen in plants, comprising treating said plants with a compositionaccording to the invention. Preferably, said composition is sprayed onthe leaves, fruits or ears of the plant. Such a method for control ofthe pathogen comprises prevention of infection and/or a decrease ofplant damage caused by said pathogen. Said method will be usable againstany pathogen mentioned above, preferably against apple scab. A furtherembodiment in the present invention is the use of a Cladosporium isolateaccording to the invention, or a composition according to the inventionfor the prevention or treatment of infection of leaves, fruits or earsof a plant by a pathogen. Further comprised in the invention is aCladosporium strain or a composition according to the invention for usein the control of a plant pathogen infection.

LEGENDS TO THE FIGURES

FIG. 1 Scab symptoms caused by Venturia inaequalis (Vi) on leaves (a)and fruit (b).

FIG. 2. Conidia production of Vi on apple leaves treated twice per weekwith Cladosporium cladosporioides H39 or R406 (approximately 2×10⁶viable spores ml⁻¹) in an organically managed orchard. Randwijk 2007.Statistically significant effects of individual treatments in comparisonwith the control treatment are indicated by ‘*’ separately per samplingdate; one-sided unprotected LSD-test; (□=0.05). N.s.: no significantdifference; n.v.: no value

FIG. 3. Predicted infection periods, dates of spray applications ofCladosporium cladosporioides H39 and sulphur, and sampling dates (L:Labelling of new leaves; S: Sampling).

FIG. 4. Effect of treatments with conidia of Cladosporiumcladosporioides H39 or sulphur on conidia production of Venturiainaequalis. Bars of the same sampling date with a common letter dodiffer significantly (LSD-test; □=0.05).

FIG. 5. Effect of treatments with conidia of Cladosporiumcladosporioides H39 or sulphur on scab severity. Bars with a commonletter do differ significantly (LSD-test; □=0.05).

DETAILED DESCRIPTION

As detailed above, apple scab is a disease with tremendous economicalimpacts for the apple growers. This is especially the case for applescab control by organic farmers, who have few or no alternative chemicalpesticides, especially now that some of the agents, which are commonlyused are tending to be phased out because of environmental and generaltoxicity risks.

An alternative is being sought in the form of extracts from plants,since plant extracts have been described to be possible candidates fortreating fungal diseases and since these would comply with therequirements for organic growing. Recently, the use of extracts fromYucca has been documented (WO 2007/139382).

However, next to plant extracts also natural occurring micro-organismsare useful in the control of plant pathogens. Especially many fungi areknown because of their biocidal effects, such as Penicillium andTrichoderma. More specifically, there also exist fungi which areespecially useful against plant pathogens. Also in the case of applescab such fungi have been discovered, such as Chaetomium globosum(Boudrau, M. A. et al., 1987, Phytopathol. 77:1470-1475),Microsphaeropsis ochracea (Carisse, O. et al., 2000, Phytopathol.90:31-37), Ophiostoma sp. and Phoma sp. (Ouimet, A., et al., 1997, Can.J. Bot. 75:632-639). General overviews may be found in Burr, T. J. etal., 1996, Biol. Control 6:151.157 and Fiss, M. et al., 2003, Z.Pflanzenkrankh. Pflanzenschutz, 110(6):513-523. Recently, it has beendisclosed (Ziedan, E. H. E., 2006, Res. J. Agric. & Biol. Sci.2(6):262-267) that an isolate of Cladosporium cladosporioides, derivedfrom the phyllosphere of grapevine, administered together with benzoicacid was effective against the grapevine downey mildew causing fungusPlasmopara viticola. Also was found that particular isolates ofCladosporium cladosporioides can be used against Botrytis and Fusariuminfections (Newhook, F. J., 1957, New Zealand Journal of Science andTechnology 23:23-54. Eden et al., 1996, Plant Pathol. 45:276-284).

In the present invention it has now been discovered that two isolates ofCladosporium cladosporioides are effective against fungal foliarpathogenic diseases, such as apple scab. These two isolates areCladosporium cladosporioides H39 and Cladosporium cladosporioides R406,deposited on 13 Dec. 2007 with the Centraal Bureau voorSchimmelcultures, Baarn, the Netherlands under nrs. CBS 122244 and CBS122243, respectively. As is shown in the experimental section of thespecification, these two new isolates had a clear effect on apple scabdevelopment, while other isolates of Cladosporium cladosporioides didnot have any effect.

Cladosporium cladosporioides (Fresen.) de Vries belongs to the group ofthe Fungi imperfecti and its growth is characterized by colonies ofabout 3 cm in diameter when grown for seven days on potato-dextrose agarat 20° C. Colonies of Cladosporium cladosporioides are effuse, olivegreen or oliviceous brow, velvety; reverse on malt agar greenish black.Conidiophores are macronematous and micronematous, sometimes up to 350μlong but generally much shorter, 2-6μ thick, pale to mid olivaceousbrown, smooth or verrucose. Ramo-conidia are 0-1 septated, up to 30μlong, 2-5μ thick, smooth or occasionally minutely verrucose. Conidia areformed in long branched chains, mostly O-septated, ellipsoidal orlimoniform, 3-11×2-5 (mostly 3-7×2-4)μ, pale olivaceous brown, mostcommonly smooth but verruculose in some strains (Ellis, M. B., 2001,Dematiaceous Hyphomycetes).

It has been reported that one of the metabolites, cladosporin, acts asantifungal. This cladosporin has been shown to have antibioticproperties (Scott, P. M. et al., 1971, J. Antibiotics 24:747-755). Yet,as already discussed above, it was found in our experiments that not allisolates of Cladosporium cladosporioides had an inhibitory effect onapple scab infection. Apparently, either cladosporin is not functionalagainst Venturia inaequalis, or, more likely, the isolates of the fungusdiffer in their phenotype of cladosporin expression and secretion.Possibly, also other mechanisms of mode-of-action are involved inantagonism of H39 and R406 towards Venturia inaequalis and other plantpathogens.

Cladosporium spp. are common epiphytic colonizers of leaf surfaces,often found in high densities (Dickinson, 1981). Epiphytic colonizationby Cladosporium spp. is also reported for apple leaves (Fiss et al,2000) and fruits (Teixido et al., 1999). Such epiphytic populations ofCladosporium spp. may contribute to naturally occurring intrinsicbiological control. In this case, conservation biological control(Eilenberg et al., 2001) protecting such beneficial populations byavoiding fungicides with side effects on non-target populations (Teixidoet al., 1999; (Walter et al., 2007) or even selectively stimulating suchnaturally occurring beneficial populations, e.g. by application specificnutrients, may contribute to scab prevention.

As is shown in the Examples, the two isolates of the present inventionshowed a clear effect in the control of apple scab. It is furthersubmitted that not only apple scab but any fungal plant pathogen ofleaves, fruits or ears can be controlled by said isolates. Said fungalfoliar pathogen is selected from the group consisting of apple scab(Venturia inaequalis), pear scab (Venturia pirina), leaf spot(Blumeriella jaapi), rose black spot (Diplocarpon rosae/Marssoninarosae), brown spot (Stemphylium vesicarium, powdery mildews of variousplants such as strawberry, roses and apple, sooty blotch (Gloeodespomigena), fly speck (Zygophalia jamaicensis), peach leaf curl (Taphrinadeformans), brown rot/spur canker (Monilia fructigena, M. laxa), pearrust (Gymnosporangium sabinae/G. fuscum), canker (Nectria galligena),rose black spot (Diplocarpon rosae), rust on roses (Phragmidiumtuberculatum/Phragmidium spp.), Botrytis spp. in various plants,Mycosphaerella brassicicola in cabbage, Mycosphaerella fijiensis inbanana, Alternaria spp. in Brassica, potato and various other plants,Fusarium spp. especially in cereals including maize, Phytophthorainfestans of potato and Plasmopara viticola in grapevine, preferablywherein said fungal foliar pathogen is apple scab.

It appeared in the case of H39 that the way of culturing and theformulation of the fungus had a marked influence on the inhibitoryeffect. Conidia of C. cladosporioides H39 produced on oat meal agar werenot effective under orchards conditions but those produced on oats in anfermenter consistently showed moderate effectivity. Microscopicalobservations showed that conidia produced on oats in an fermenter werelarger than those produced on oat meal agar. The effect of growth mediumon the spore quality of antagonists resulting in a better performanceunder environmental stress conditions has been demonstrated for severalother antipathogenic fungi, e.g. cells of Candida sake produced onlow-a_(w)-modified media (Teixido et al., 1999). Colonization of appleleaves by introduced fungi depends on the biology of the introducedspecies (Kinkel et al., 1989). In their case study, introducedpopulations of C. globosum significantly decreased within a few days incontrast to populations of A. pullulans. In the below experiments, theinhibitory compositions were applied at 3 to 4 days interval toguarantee high populations level on the leaves. However, first resultson population dynamics of Cladosporium cladosporioides indicate thatafter application of H39 such populations increased and that suchtreatment effects were found even after several weeks (see example 2).

Formulation of the fungus is important for the viability of the fungalspores contained in the composition that is applied to the plant. It hasappeared that a formulation wherein glucose is used as carrier for theCladosporium spores performs better than unformulated fungus.

Alternatively, an antifungal composition of the present invention maycontain an extract of one or both of the Cladosporium isolates of theinvention. Extracts of the two isolates can be obtained as described inthe literature for other Cladosporium extracts, and can be readilyobtained by a person of skill in the art (see e.g. Ding, L. et al.,2008, Current Microbiol., 56:229-235).

Further, the composition may contain additional compounds that arecommonly used in spraying solutions, such as preservatives, surfaceactive compounds, wetting agents, and the like. It is also possible toinclude further antifungal compounds or organisms in the composition toenhance the inhibitory effect. In such a case, lower levels oftraditional chemical fungicides can be used, thereby enabling a desiredreduction in the amount of fungicides. As discussed above, the chemicalfungicides reduce the growth of the plant pathogenic fungus, and thuswill allow for a prolonged opportunity for the Cladosporium to achieveits effect. It is thus submitted that a combination of the isolates ofthe present invention with suitable chemical fungicides willsynergistically inhibit the fungal foliar pathogen thereby exceedingbeyond a simple addition of the individual effects. Also a combinationof the isolates of the invention with any other microbial fungal foliarpathogen antagonist will be useful in the present invention.

Another embodiment of the invention is a method to control a fungalpathogen on leaves, fruits and ears, comprising treating said plantswith a composition according to the invention.

In such a method plants are sprayed with a composition according to theinvention before or after an infection with the plant foliar pathogen.Spraying before infection will be useful to prevent an infection.Although, if the spores of the isolates are not completely viable, suchanaphylactic spraying should be repeated regularly. If the spores aresufficiently viable, the Cladosporium isolate will be able to grow inthe phyllosphere of the plant, thus providing a long-lasting protectionby preventing infections and reducing pathogen sporulation, thus slowingdown epidemics. Furthermore, the Cladosporium isolate will reduceascospore production in overwintering apple leaves and of spores ofother plant pathogens in their residues which will result in lessdisease in the following season.

Once an infection has occurred, spraying should be performed regularlyin order to effective control the disease.

Treatment with a composition according to the invention can be done bothpre-harvest, i.e. when the fruits are still on the plant, but alsopost-harvest. It is shown in the experimental part that alsopost-harvest treatment is effective to combat fungal infections.

Yet another embodiment of the invention is the use of a Cladosporiumstrain according to the invention, or a composition according to theinvention for the prevention or treatment of infection by a fungalfoliar pathogen.

A further embodiment of the present invention is a Cladosporium strainas identified above, or a composition according to the invention for usein the control of a fungal foliar pathogen infection.

EXAMPLES Example 1 In Vitro and Field Tests 2006

Material and Methods

Collection of Candidate Antagonists

Scab infected apples leaves were collected in September 2004 at 216locations in The Netherlands, Belgium, and northwest and centralGermany. Sampling locations were recorded by GPS. Most samples,consisting of 10-50 leaves, originated from old standard tress orabandoned orchards without any cropping management. From each sample,green leaves with sporulating colonies of V. inaequalis were incubatedfor 3 days at 20° C. in moist chambers. Thereafter, V. inaequaliscolonies were visually inspected at 10 to 60× magnification fordevelopment of fungi different from V. inaequalis. Fungal mycelium orspores were isolated from the aerial parts of the colony with a sterileneedle and transferred to oat meal agar (20 g oat meal, 15 g agar, 1000ml tap water) and V8 agar (200 ml V8 juice, 3 g CaCO₃, 20 g agar, 1000ml tap water), both containing 100 mg l⁻¹ streptomycine and 15 mg l⁻¹tetracycline. Pure cultures were prepared from the developing colonies.

Pre-Screening of Candidate Antagonists

A rapid throughput system was used for a first check of candidateantagonists regarding their potential risks and economical feasibilityof the development of a biocontrol product. Fungi belonging to thegenera Aspergillus, Penicillium or Fusarium were discarded because ofthe potential of various species within these genera to producemycotoxins. The remaining isolates of hyphal fungi were cultured inPetri dishes on oat meal agar for 21 days at 18° C. and 12 h per dayblacklight; yeasts were cultured on basal yeast agar (10 gbacteriological peptone, 1 g yeast extract, 20 g glucose, 20 g agar,1000 ml tap water) for 5 days at 18° C. For each isolate, the productionof spores or yeast cells per plate was determined with the aid of ahaemocytometer after preparation of suspensions using sterile tap watercontaining 0.01% Tween 80. Isolates producing less than 1×10⁵ spores oryeast cells per plate were discarded. Ten μl of the suspensions wereplated in duplicate in sterile wells, 16 mm in diameter containing 1.5ml malt agar (1 g malt extract, 15 g agar, 1000 ml tap water). Differentplates with inoculated wells were incubated at 5, 18 and 36° C. in thedark for 14 days. An additional plate with wells containing malt agaradjusted to approximately −10 and −7 MPa by adding KCl₂ were incubatedat 18° C. for 14 days. Wells were inspected for fungal growth and fungiproducing colonies at 36° C. and those not producing colonies at 5° C.or at −10 or −7 MPa were discarded. Remaining isolates were stored onpotatoe dextrose agar (Oxoid, 39 g, 1000 ml tap water) at 4° C. untilfurther use.

Production of Fungal Inocula

Conidia of the monospore isolate MB 363B of V. inaequalis obtained fromM. Bengtsson, Royal Veterinary and Agricultural University,Frederiksberg C, Denmark, were produced following the method of Williams(1976). Duran bottles (500 ml) with 40 ml potato dextrose broth (24 gl⁻¹) and a wick of cheese cloth sized 10×25 cm spread on the innersurface of the bottle, the lower part touching the medium, wereautoclaved and inoculated with 250 □l of a suspension of mycelialfragments of V. inaequalis, prepared by flooding a culture of the fungusgrown on potatoe dextrose agar with sterile water and gently rubbingwith a sterile rubber spatula. Inoculated bottles were incubated at 18°C. in the dark. During the first 4 days, bottles were incubated athorizontal position so that most of the wick was covered by the broth,thereafter bottles were incubated in an upright position. Bottles werecarefully rolled at day 2, 7 and 11 to distribute the broth with growingmycelium on the entire surface of the wick. After 14 days, the nutrientbroth was decanted and 200 ml sterile tap water was added and thebottles were shaken thoroughly by hand for several minutes. Theresulting conidial suspension was filtered through sterile nylon gauzewith a mesh of 200 □m and centrifuged at 5800 g for 30 min at 6° C. Thepellet with conidia was re-suspended in tap water; the conidialconcentration was determined with the aid of a haemocytometer andadjusted to 5×10⁵ spores ml⁻¹ by adding sterile tap water. Thesuspension was stored at −18° C. until use. The yield was approximately3×10⁶ conidia per bottle.

For the screening experiments, candidate isolates of hyphal fungi weregrown on oat meal agar for 28 days at 18° C. and 12 h blacklight perday; yeasts were grown on basal yeast agar for 5 days at 18° C.Suspensions of spores or yeast cells were prepared by flooding cultureswith sterile tap water containing 0.01% Tween 80, gently rubbing with asterile rubber spatula and filtration through sterile nylon gauze with amesh of 200 □m. Concentrations of the suspensions were determined withthe aid of a haemocytometer and concentrations were adjusted to 1×10⁶spores or cell ml⁻¹ by adding sterile tap water containing 0.01% Tween80.

For applications in the orchards, fungi were produced in the same waybut concentrations were adjusted to 2×10⁶ spores ml⁻¹. Conidia of 2isolates were also produced in a fermenter, dried and formulated aswettable powder and stored at 5° C. Viability of dried conidia wasdetermined before the beginning of the field experiments on agar. Theconcentration of conidial suspensions was adjusted to 2×10⁶ viableconidia ml⁻¹. Viability of spores in the suspensions applied to thefield was always checked again by spraying suspension on malt extractagar (1 g malt extract per liter) amended with tetracycline at 15 mg l⁻¹and streptomycine at 100 mg l⁻¹ in the orchard and assessing thepercentage of germinated spores after incubation at 20° C. for 24 h.

Seedling Assay

Seeds of apple cv Golden Delicious (G.J. Steingaesser & Comp. GmbH,Miltenberg, Germany) were seeded in moist sand and stratified at 4° C.for 6 weeks in the dark. Thereafter, seeds were further grown in themoist sand at room temperature and daylight. After approximately 14days, young seedlings were transplanted into potting soil, one seedlingper pot (6 cm square, 8 cm height). Seedlings were grown for 28 dayswith cycles of 16 h light at 18° C. and 8 h dark at 12° C. Plants usedin experiments had at least 4 fully expanded leaves.

Seedlings were sprayed with conidial suspensions of V. inaequalis (1×10⁵ml⁻¹) until run-off and placed in a moist chamber consisting of aplastic trays closed by a transparent plastic top. After 2 daysincubation at 15° C. with diffuse light, tops were removed from thetrays and seedlings further incubated for 3 or 4 days at 85% RH, 15° C.and 16 h light per day. Thereafter, V. inaequalis-inoculated seedlingswere sprayed with suspensions of the above found isolates or watercontaining 0.01% Tween 80 as control. Two seedlings were used for eachreplicate of each treatment. The sets of 2 inoculated seedlings wereplaced in a polyethylene tent in a block design with 6 blocks(replicates) and complete randomization within blocks. Touching ofleaves of neighbouring plants or the polyethylene was avoided. Seedlingswere grown for 9 to 12 days at 15° C., with 16 h light per day at 138 □Es⁻¹ m⁻².

From both seedlings of each replicate, the lowest 5 true leaves(experiments 1-5) or the youngest just unfolded leaf at the beginning ofthe experiment (labelled with a metal ring; ‘young leaves’) and the 3next elder leaves (experiments 6-14; ‘old leaves’) were carefullyremoved. Leaves of each replicate set of leaves were pooled, put intoDuran bottles (100 ml) containing 15 ml of tap water with 0.01% Tween 80for samples containing 2 youngest leaves or 30 ml for samples ofcontaining 10 (exp 1-5) or 6 (experiments 6-14) leaves. Bottles wereshaken with a flask shaker at 700 OCS min⁻¹.

Concentration of conidia of V. inaequalis was determined for eachsuspension with the aid of a haemocytometer. Leaf surfaces of eachreplicate set of leaves were measured with an area meter (Li-CORBiosciences, model 3100, Lincoln, U.S.A.).

Orchard Assay

Several rows of var. Jonagold within an organically managed orchard atRandwijk, The Netherlands, were pruned during spring and summer 2006 sothat trees produced new shoots with young leaves highly susceptible forV. inaequalis. Depending on tree development and growth conditions forthe trees pruning was carried out approximately 3 to 5 weeks before aset of trees was used for an experiment. Also the majority of youngfruits were removed from the trees to stimulate the development of newshoots. Trees used for experiments in early summer (experiments 1 to 4)were not treated for scab control in 2006. Trees used in laterexperiments after half of July were treated with a sulphur schedule ascommon for the organic orchard in the early season to slow down the scabepidemic but remained untreated at least 3 weeks before and during theexperiments.

A series of 8 experiments was carried out, each on a different set oftrees. For each experiment 2 to 6 trees, depending on the number ofnewly produced shoots per tree, were chosen for each of 6 blocks(replicates). Within each block, 7 treatments were carried out. For eachtreatment (per replicate) 3 shoots were used which were produced on thesame branch. Shoots were labelled with coloured metal rings so that the2 youngest leaves fully expanded at the day of the first treatment couldlater be distinguished from the other leaves of the shoot. Treatmentsconsisted of spraying tap water containing 0.01% Tween 80 as control, orsuspensions of freshly produced spores of the antagonist C.cladosporioides H39. Separate treatments were carried out withfermenter-produced spores formulated as dry powders and resuspended intap water containing 0.01% TWEEN 80. Spray applications were done usinga compressed air-driven knapsack sprayer at 250 kPa until run-off. Thedifferent experiments were carried out in the period between Jun. 22 andSep. 28, 2006. Experiments started with the first treatment 1 to 3 daysafter an infection period for V. inaequalis had been predicted accordingto the Mills table based on leaf wetness duration and temperature.Starting dates for the 8 experiments were Jun. 22, Jun. 29, Jul. 6, Jul.31, Aug. 3, Aug. 7, Aug. 21, Aug. 24, and Aug. 24, 2006. In experiment 8the period between expected infection period and first application wasprolonged to 10 days, but leaves were labelled already 1 to 3 days afterthe expected infection period as in the other experiments. During allexperiments, subsequent treatments were carried out at 3 to 4 dayintervals. In the first 3 experiment, leaves were sampled 18 days afterthe first treatment (and thus 19 to 21 days after an expected infectionperiod). In experiments 4 to 7, leaves were sampled 24 to 25 days afterthe first treatment (and thus 25 to 28 days after an expected infectionperiod) to increase the time period for V. inaequalis sporulation andfor possible antagonistic interactions. In experiment 8, leaves weresampled 24 days after the first application and thus 34 days after theexpected infection period.

In each experiment, the 2 youngest leaves fully expanded at thebeginning at the experiment together with the 2 next younger leaves(expanded during the course of the experiments) were pooled for the 3shoots belonging to the same replicate so that a sample consisted of 12leaves (‘young leaves’). From the same shoot, the next elder 3 to 12leaves, depending on shoot size, were also sampled and pooled so thatsamples consisted of 9 to 36 leaves (‘elder leaves’). The average numberof elder leaves sampled in the different experiments was 21.8. Samplesof young leaves were put into 250-ml glass bottles. Within 2 h, 100-150ml (depending on amount of leaves) of tap water with 0.01% Tween 80 wasadded and bottles were shaken with a flask shaker at 700 OCS min⁻¹ for10 min. Samples of older leaves were processed in the same way using1000-ml plastic bottles with 150-350 ml (depending on number of leaves)of tap water added before shaking. From the obtained suspensions,sub-samples of 6 ml were stored at −18° C. The concentration of conidiaof V. inaequalis was determined for each suspension with the aid of ahaemocytometer. The leaf surface of all leaves per sample was measuredwith an area meter.

Statistics

The number of V. inaequalis conidia produced cm⁻² leaf was calculatedper replicate. If no conidia were detected, a detection limit was set atone conidium counted in the conidial suspension, resulting in an averagedetection limit for the various experiments of approximately 100 conidiacm⁻², e.g. ranging, depending on the leaf surfaces for the variousreplicates, between 74 and 212 cm⁻² for the first 3 experimentsconducted under controlled conditions. Data obtained for 2 differentleaf ages were natural logarithmic-transformed and analysed separatelyper leaf age by ANOVA. Experiments conducted under controlled conditionswere analysed separately by comparing means of the control treatmentsand the individual antagonist treatments using unprotected LSD-tests(□=0.05). Since P-values of ANOVAs often were greater than P=0.05, nofurther multiple comparisons between antagonist treatments were made(Ott and Longnecker, 2001). For the field experiments, naturallogarithmic-transformed data obtained for 2 different leaf ages wereanalysed separately per leaf age by ANOVA with a block design, withindividual experiments considered as main blocks and treatments wererandomized within the 6 replicate blocks of each experiment.Statistically significant treatment effects (protected LSD-tests;□=0.05) were indicated.

Results

Isolation of Fungi from V. inaequalis Colonies and Pre-Screening

Growth of fungi different from V. inaequalis was frequently observed insporulating colonies of the pathogen and several hundreds of fungalisolates were obtained. Isolates were grouped according to colonyappearance and in total 148 isolates representing various colony typeswere tested in the pre-screening. 131 out of the 148 isolates producedmore than 1×10⁵ spores or yeast cells per plate and grown at differenttemperatures and water potentials. From these remaining 131 isolates,all were able to grow at 5° C.; 16 did not grow at −10 MP. 14 grew at36° C. of which 1 isolate did not grow at WP=−10 MPa. Since many of suchisolates with the preferred combination of characteristics belonged toCladosporium spp., only a sub-set of randomly chosen isolates of thisgroup was tested on seedlings. In total, 63 out of the 102 isolates werefurther tested in bio assays on apple seedlings.

Seedlings Tests

Fourteen experiments on seedlings were carried out. In the controltreatments of the first 5 experiments, the conidia production on thefive youngest leaves of the seedling was on average 3596 conidia cm⁻²and ranged for the different experiments between 1339 and 10509 conidiacm⁻² (backtransformed values). Since conidia production was high on theyoungest leaf and much less on the elder leaves it was decided to samplesuch leaves separately in the subsequent experiments. In experiment6-14, average conidiation on the young leaf was 2896 conidia cm⁻² andranged for the different experiments between 728 and 6186 conidia cm⁻²(backtransformed values). For elder leaves, average conidiation was 453conidia cm⁻² and ranged for the different experiments between 144 and1313 conidia cm⁻² (backtransformed values). Variation in conidiation wasnot only high between experiments but also within experiments betweensets of replicate seedlings.

Most of the 80 candidate isolates tested on seedlings did notstatistically significantly reduce conidiation of V. inaequalis. Oneisolate, C. cladosporioides H39 caused a significant reduction of V.inaequalis on the young or elder leaves which could be repeated insubsequent independent experiments. However, efficacies (calculated onbase of backtransformed values for young leaves) of this antagonistvaried between 55 to 79% for C. cladosporioides H39 in the experimentsin which it was tested and conidiation reduction was in some cases notstatistically significant (Table 1). A few more isolates showed a strongstatistically significant antagonistic effect in one experiment but sucheffects could not be repeated. Such isolates belonged to A. pullulans,C. cladosporioides, P. pinodella, and Cladosporium spp. In no casesignificant enhancements of conidiation of V. inaequalis afterapplication of candidate isolates was observed.

Orchard Testing 2006

Apple scab developed moderately in the orchard before the firstexperiment started at 22 June. In June and first half of July, dryconditions accompanied with day temperatures often above 30° C. were notfavourable for further apple scab development. Thereafter, rainy andcold weather was strongly favourable for apple scab until the end of theexperiments.

During the first 3 experiments conducted under dry and warm conditions,2.1 to 12.8×1000 conidia cm⁻² (backtransformed values) were found onyoung leaves and 9.5 to 40.3×1000 conidia cm⁻² on elder leaves on watertreated control plots (Table 2 A, B). After weather had changed to moistand cold conditions half of July, 46.3×1000 conidia cm⁻² on young leavesand 9.8×1000 conidia cm⁻² were found on leaves from control treatmentsin experiment 4. During the subsequent experiments conidia numbersincreased to 227.3×1000 conidia cm⁻² on young leaves and 132.1 conidiacm⁻² on elder leaves in experiment 8.

Viability of spores applied at the 25 application dates during thecourse of the series of experiments was assessed for each suspension onone agar plate sprayed in the field. Viability of Cladosporium sp. H39was 92% (ranging from 82 to 99%). Formulated spores of C.cladosporioides H39 had a viability of approximately 47% at thebeginning of the experiments. When spore suspensions were prepared forfield applications, formulated spores of C. cladosporioides H39 formedclusters consisting of approximately 5 to 50 spores in the sprayingsuspensions so that a precise determination of spore germination was notpossible. The majority of clusters of suspensions sprayed in the 11applications showed fungal growth. Only few spore clusters appliedbetween 10 August and 29 August showed fungal growth. It can be assumedthat stored formulated spores had been used which lost their vigourduring storage for the last treatments during experiment 4 and alltreatments during experiments 5 and 6. From end of August onwards, a newbatch of formulated spores was used of which the majority of sporeclusters showed fungal growth. No significant treatments effects werefound when data were analysed for each experiment separately. For anoverall analysis, experiments 1, 5 and 6 were excluded because viableformulated C. cladosporioides H39 was not available during experiment 1and viability was low during these experiments 5 and 6. A significantreduction of the number of conidia cm⁻² was found for treatments withformulated C. cladosporioides H39 on both young and elder leaves (Table2 B). On average, conidia production was reduced by 42% on young and 38%on elder leaves. Such a trend was found during the first 3 experimentsconducted under low disease pressure as well as during the last 2experiments conducted under extremely high disease pressure. Thenonformulated spores of the isolate did not reduce conidiation of V.inaequalis. When the formulation of C. cladosporioides H39 was appliedcontaining only a few or even no living conidia, during experiments 5and 6, no trend was found that the formulation itself had any reducingeffect on conidiation (Table 2 A).

Example 2 Orchard Test 2007

Material and Methods

Fungal inoculum

Cladosporium cladosporioides H39 was produced as in Example 1. Theformulated product contained 2.0×10⁹ spores g⁻¹ with a viability of 20%.During the experiment, the batch of formulated H39 was stored at 4° C.in the dark in plastic bags. Suspensions were made by adding theformulated granules to Tween water (0.01%) and stirring without anyfurther pre-treatments. The final concentration was 1×10⁷ spores ml⁻¹,equivalent to 2×10⁶ viable spores ml⁻¹.

Spores of Cladosporium cladosporioides R406 were produced in Petridishes on oat meal agar (20 g oat meal, 15 g agar, 1000 ml tap water) at20° C. with 12 hrs blacklight per day. After 2 weeks of incubation,spores were suspended in tap water containing 0.01% Tween 80 and addedto the plates. The suspension was filtered through gauze (200 □m mesh)to remove mycelial cells and the number of spores was counted using ahaemocytometer. Spore yields were 8-30×10⁸ spores per agar plate (90 mmdiameter) with an average viability of 93%, ranging between 73 to 97%for suspensions prepared at different dates. Viability of spores in thesuspensions applied to the field was always checked by sprayingsuspension on malt extract agar (1 g malt extract, 15 g agar 1000 ml tapwater, amended with tetracycline at 15 mg l⁻¹ and streptomycine at 100mg l⁻¹) in the orchard and assessing the percentage of germinated sporesafter incubation at 18° C. for 24 h.

Orchard

The experiment was carried out in 3 rows of var. Jonagold within anorganically managed orchard at Randwijk, the Netherlands. The aim of theexperiment was to control the summer epidemic of Venturia inaequalis(Vi) by antagonist applications starting end of June. Therefore it wasessential to allow an initiation of a mild to moderate epidemic in theorchards during the primary season. The timing of sulphur applicationswas foreseen in a way that primary infections by ascospores could occurbut progression of the epidemic could be controlled. No applications ofsulphur were planned later than end of May. Unusual weather conditionsduring the primary season of 2007 with a period of 4 weeks without anyrain were very unfavourable for Vi ascospore release and infection.After weather became more conducive after April, a mild apple scabepidemic developed until beginning of the experiment. No sulphurtreatments were carried out to reduce the progression of the epidemic.

Experimental Design, Treatments and Assessments

The experiment was carried out in a design with 6 blocks, with 2 blocksin each of the 3 tree rows. Each block consisted of 4 plots, each with 4trees. Between plots, 2 untreated trees served as buffer. The differenttreatments were randomly allocated to such plots. It was planned tospray the 4 trees of each plot at a rate of 2 l per plot twice per weekwith the following treatments: (1) tap water amended with Tween 80(0.01%) as control; (2) suspension of formulated H39 (2×10⁶ sporesml⁻¹); (3) suspension of formulated R406 (2×10⁶ spores ml⁻¹).Applications were carried out twice per week at 16 dates between 28 Juneand 20 August using a hand-held sprayer operating with compressed air(AZO, Edecon, Ede, The Netherlands) with a 2-m boom and one nozzle(Birchmeier helico saphir 1.2, 2F-0.6; pressure 250 kPa). Sinceformulated spores of R406 were not available at the beginning of theexperiment, treatment (3) was carried out with spores produced freshlyin the laboratory for each application. Applications of R406 twice perweek started with a delay at 12 July so that this fungus was appliedonly at 12 dates. Because no sufficient spores could be produced, onlyselected and labelled twigs obtained multiple sprays instead of wholetree treatments.

Assessments

Conidia production. Conidia production was assessed on susceptible youngleaves developed during the course of the experiment at 3 sampling datesfor leaves treated with Tween-water as control, or conidial suspensionsof H39 and for 2 sampling dates for R406. Sampling dates were chosen sothat sets of susceptible leaves present during a predicted infectionperiod were sampled approximately 5 weeks after the infection period.The Mills table based on leaf wetness duration and temperature was usedto predict infection periods. For each sampling, the second youngestjust unfolded leaf was labelled 1 to 3 days after a predicted infectionperiod on a set of 3 twigs belonging to the same tree in each plot.After 5 weeks, the 2 leaves just unfolded at the date of labelling andthe next 2 younger leaves, unfolded after labelling, were sampledresulting in a sample consisting of 12 leaves per plot. Leaf sampleswere put into 250-ml glass bottles. Within 2 h, 100-150 ml (depending onamount of leaves) of tap water with 0.01% Tween 80 was added and bottleswere shaken with a flask shaker (Stuart Scientific SF1, UK) at 700 OCSmin⁻¹ for 10 min. From the obtained suspensions, sub-samples of 6 mlwere stored at −18° C. and the concentration of conidia of Vi wasdetermined later for each suspension with the aid of a haemocytometer.The leaf surface of all leaves per sample was measured with an areameter (Li-COR Biosciences, model 3100, Lincoln, U.S.A.).

Sampling dates were 6 August (of leaves labelled at 2 July), 16 August(of leaves labelled at 12 July), and 20 August (of leaves labelled at 16July).

Epiphytic and endophytic colonisation. Twenty labelled leaves of treestreated with Tween-water (control) or spore suspensions of H39 and oftwigs treated with spore suspensions of R406 were collected at 4 Octoberfrom each plot. All sampled leaves had been developed on the treesduring the period between 28 June and 20 August when the series sprayapplications had been carried out. It thus can be assumed that allleaves obtained one or several spore applications at young developmentalstages.

From each leaf, one leaf disc (9 mm diameter) was cut using acorkbohrer. The resulting 20 leaf discs per sample were pooled insterile 50-ml vials containing 10 ml of sterile Tween-water (0.01%) andvials were shaken using a flask shaker at high speed (700 OCS min⁻¹) for10 min. From the resulting suspensions, serial dilutions (1:10) wereprepared and 100 □l of undiluted and diluted suspensions were plated oneach of 2 agar plates containing malt agar (10 g malt extract, 15 gagar, 1000 ml tap water) amended with 100 mg l⁻¹ streptomycine and 15 mgl⁻¹ tetracycline. Plates were incubated at 20° C. in the dark andcolonies of Cladosporium spp., other hyphal fungi and yeasts werecounted after 4, 7 and 11 days. From the colony counts, the number ofcolony forming units (CFU) cm⁻² leaf surface was calculated for eachsample. After shaking of the leaf disks to remove epiphytically presentfungi, the pooled 20 leaf disks per sample were surface-sterilised andhomogenated in 5 ml sterile water using a mortar. For surfacesterilisation, leaf discs were dipped in 96% ethanol followed bysubmersion for 1 minute in 0.5% sodium hypochlorite and subsequentlyrinsed 3 times with sterile water. From the resulting homogenates serialdilutions (1:10) were prepared in sterile water and 100 □l of undilutedand diluted suspensions were plated on each of 2 malt agar platescontaining 100 mg l⁻¹ streptomycine and 15 mg l⁻¹ tetracycline. Plateswere incubated and colonies of endophytic fungi were assessed asdescribed above.

Statistics

The number of Vi conidia produced per cm⁻² leaf surface was calculatedper replicate sample. Natural logarithmic-transformed values of thecontrol treatment were compared with the individual antagonisttreatments separately for both leaf ages by one-sided unprotectedLSD-test (□=0.05). The logarithmic-transformed numbers of CFU per cm⁻²leaf surface were analysed by ANOVA followed by two-sided LSD-tests(□=0.05).

Results

Weather conditions during the period of the experiments with frequentrainfalls and moderate temperatures were very favourable for apple scab.At the beginning of the experiments only light symptoms were present inthe orchards. At the end heavy scab symptoms were observed on leaves oftrees within the experimental plots and in the neighbourhood.

Spores of R406 produced freshly for each application date had highgermination rates, generally above 80% (Table 3). Formulated spores ofH39 germinated for 20-30% during the first weeks of the experiment butgermination dropped to 4-16% for the last 4 treatments. The exceptionalhigh value for spore germination of 76% at the first spraying date canbe explained by experimental variation due to the small sample size ofonly 1 plate sprayed under orchard conditions.

On leaves sampled at 6 August, 16 August and 20 August fromwater-treated trees, 77,200 (13,900 to 139,800), 45,300 (35,200 to55,200) and 32,200 (8,800 to 111,800) conidia cm⁻² were produced onaverage (backtransformed means, range for 6 replicates in brackets; FIG.2). Variation between replicates thus was considerably high. On leavesof trees treated with H39, the number of spores was statisticallysignificantly lower with 24,200 conidia cm⁻² (69% reduction) at thefirst sampling date and 22,000 conidia cm⁻² (51% reduction) at thesecond sampling date. For the last sampling date, no treatment effectwas observed for H39. For the sampling dates when leaves treated withR406 were available, conidia counts at 16 August were 24,300 conidiacm⁻² for leaves treated with R406 resulting in a statisticallysignificant reduction of conidia by 46%. For 20 August, only a reductionby 15% was observed and counts did not differ from the controltreatment.

No treatment effects on leaf development or symptoms of phytotoxicitywere observed.

Close to leaf fall, the endo- and epiphytical colonisation of leaves wasassessed by plating leaf washings and homogenates of surface sterilisedleaves on agar and counting of colony forming units (CFU). Theendophytic colonisation by Cladosporium spp. was significantly higherand epiphytical colonisation by Cladosporium spp. tended to be higherfor leaves which had been treated with H39 during summer. The endo- andepiphytical colonisation of H39-treated leaves by other hyphal fungitended to be lower compared to untreated leaves.

Epiphytical colonisation by yeasts was significantly lower forH39-treated leaves.

Example 3 Field Test 2005/2006

Materials and Methods

Experimental Design and Treatments

In autumn of 2005, 25 fungal isolates belonging to different speciesincluding Cladosporium cladosporioides R406 were applied on apple leavesfrom an organically managed orchard in Randwijk, the Netherlands, andtreated leaves were placed on the orchard floor until next spring. Theselection of candidates was based on the results of the pre-screeningcarried out on economical feasibility and possible risks (see example1). Spores of isolate R406 were produced on oat meal agar (28 daysincubation at 18° C.). Spore suspensions (500 ml per isolate) wereprepared by flooding cultures on agar with sterile water containing0.01% Tween 80. After gently rubbing with a rubber spatula to removespores from fungal cultures, suspensions were filtered through sterilenylon gauze with a mesh of 200 □m. Concentrations of suspensions weredetermined with the help of a haemocytometer and adjusted to 2×10⁶spores ml⁻¹. Spray applications were carried out using hand heldsprayers operating with pressed air. Leaves of extension shoots werecollected from the trees at 17 October to carry out the sprayapplications under controlled conditions in a glasshouse compartment.During the period between picking the leaves and fixing leaves innettings again on the orchard floor, leaves were stored at 5° C. Leaveswere fixed in nettings, each with 40 randomly chosen leaves, at 18 and19 October. At 20 October 4 nettings with leaves per replicate of eachtreatment were sprayed with spore suspensions or water withapproximately 20 ml of the suspensions (or water in the controltreatments) at both sides. Two of the nettings, each containing 40leaves, were destined for determination of the potential ascosporeproduction of V. inaequalis, the other two nettings for determination ofV. inaequalis population densities by TaqMan-PCR. After applications,leaves were dry again within approximately 30 min. Nettings with treatedleaves were fixed on the orchard floor at 21 October on bare soil withinthe organic orchard at Randwijk. There were 6 blocks (replicates) indifferent rows of the orchard.

Each spore suspension was also sprayed on an agar plate ( 1/10 maltagar). Plates were incubated at 18° C. for 2 days and germination ratewas determined. Spores of R406 had a viability of >90%.

Assessments

At 23, 24, 25, 30 and 31 Jan. 2006, respectively the nettings of thereplicates 1 to 6 destined for the quantification of the V. inaequalispopulation densities by TaqMan-PCR were collected in the orchard,carefully rinsed with tap water to remove adhering soil and allowed todry overnight at room temperature. Leaf residues of the 2 nettings perreplicate of each treatment were removed from the nettings, cut in 1- to2-cm pieces and a sample of 5-10 g was freeze dried and subsequentlypulverized in a laboratory mill with a 1-mm mash sieve. Powdered sampleswere stored at −18° C. DNA was extracted and DNA of V. inaequalis wasquantified by species-specific realtime PCR (TaqMan-PCR).

At 20 March (block A), 22 March (block B), 27 March (block C), 29 March(block D), 3 April (block E), and 5 April (block F), the nettings werecollected in the orchard to quantify the potential ascospore productionafter ascospore extraction by bubbling air in water. Leaf residues ofthe 2 nettings per replicate of each treatment were air-dried (20° C.,70% RH, 2 days) and weight was determined. Subsequently, a sub-sample ofmaximum 7-17 g air-dried leaf residues of each sample was spread in aplastic tray on moist filter paper. Trays were covered by a plastic bagand leaf residues were incubated in these moist chambers for 7 days at20° C. in the dark followed by 14 days at 20° C. with 12 h light per dayto allow maturation (of a substantial fraction) of asci.

Different climate rooms were used for incubation but leaves belonging tothe same block were always incubated in the same room.

After incubation, leaf residues were transferred into 1000 ml-plasticbottles containing 150-350 ml water depending on the amount of leafresidues. Air (250 l per h) was bubbled through the water resulting inheavy turbulence during 2 h. Thereafter, the resulting suspension waspassed through a sieve (1 mm mesh) to remove leaf debris. Twosub-samples (8 ml) of the suspension were stored at −20° C. Ascosporeconcentration in the suspensions was determined microscopically using ahaemocytometer. Ascospore production was expressed as production per 80leaves (originally fixed on the orchard floor in autumn) or per gair-dried leaf residue present at sampling date in spring. The ascosporeproduction per 80 leaves is a result of the possible effect of thetreatment on the decomposition and the ascospore production.

Statistics

Data obtained for ascospore numbers were logarithmic-transformed(log₁₀). All data were analysed by ANOVA. Means of the controltreatments and the individual antagonist treatments were compared usingLSD-tests (□=0.05). Since P-values of ANOVAs often were greater thanP=0.05, no further multiple comparisons between antagonist treatmentswere made.

Results

The amount of DNA of V. inaequalis was quantified in leaf residuessampled end of January 2006 by realtime PCR (TaqMan-PCR) using aspecies-specific primer pair and probe. The total amount of V.inaequalis-DNA in the total residues left from 80 leaves was calculated.Residues of leaves treated by Cladosporium cladosporioides R406 hadsignificantly less V. inaequalis-DNA compared to the untreated control(Table 6).

The number of ascospores produced on leaves in spring was high with onaverage 7.5×10⁵ (back-transformed values) for the residues of 80 leavesfrom the control treatment (Table 7). Per g leaf residue, 3.4×10⁴ascospores were produced in the control treatment (Table 8). Forresidues of leaves treated with isolate Cladosporium cladosporioidesR406 ascospore counts were approximately 70% lower. These differencesbetween control treatment and treatment with R406 were statisticallysignificantly for both the number of ascospores produced on the residuesof 80 leaves and the number of ascospores produced per g of leaf residue(Tables 7 & 8). No other treatment with different candidate antagoniststended to cause a reduction of ascospores.

Example 4 Effect of Cladosporium cladosporioides H39 on ConidiaProduction of Venturia inaequalis Under Orchard Conditions

Objective

Applications of conidia of the antagonist C. cladosporioides H39,pilot-formulated as water dispersible granule (WG), reduced the conidiaproduction of the apple scab pathogen Venturia inaequalis under orchardconditions in experiments carried out in 2006 and 2007 conditions (seeExample 1 and 2). The objective of the experiment carried out in 2008was to confirm these results in another season and to obtain firstinsight in the best timing of antagonist applications after predictedinfection periods for V. inaequalis.

Production of Conidia of H39

For applications in the orchard, conidia of C. cladosporioides H39 wereproduced in a Solid-State Fermentation (SSF) system by PROPHYTABiologischer Pflanzenschutz GmbH, Germany. The harvested conidia wereformulated as a water dispersible granule (WG). For the preservation ofproduct quality, the final product was stored at 4° C. Viability ofdried conidia was determined on malt extract agar (1 g malt extract l⁻¹)before the beginning of the field experiment. Conidia incubated for 24hours at 20° C. with germ tubes longer than half of the minimum diameterof a conidium were considered to be viable.

Orchard Assay

The experiment was carried out in the organically managed orchard atApplied Plant Research, Randwijk, The Netherlands on 8 years-old treescv Jonagold. The aim of the experiment was to control the summerepidemic of apple scab by antagonist applications. Therefore it wasessential to allow an initiation of an epidemic in the orchard duringthe primary season. Weather conditions during the primary season of 2008favoured scab development and many scab symptoms were found in theorchard beginning of June. A severe hail event seriously damaged theorchard at June 22. At June 24 and 26, Topsin-M (a.i. 500 g thiophanatemethyl l⁻¹, Certis Europe B. V., Maarsen, The Netherlands) was appliedat of rate of 140 ml per 100 liter water (applied at 1000 l ha⁻¹) in theentire orchard and the neighbouring orchards to prevent wound infectionsby European fruit tree canker (Nectria galligena). No further fungicidetreatments were carried out to reduce the progression of the scabepidemic before or during the experiment. During the following weeks,trees produced abundant new shoots with new leaves. Such newly formedleaves, not damaged by hail or reached by the fungicide sprays, wereused in the experiment.

The experiment was arranged in a design with 6 blocks, with 2 blocks inthe same tree row. Each block consisted of 3 plots, each with 4 trees.Between plots, 2 untreated trees served as buffer. The following 3different treatments were randomly allocated to the plots: (1) untreatedas control; (2) conidial suspension of formulated H39 (2×10⁶ viableconidia ml⁻¹; 2 l per plot). The spray additive Trifolio S-forte(Trifolio-M GmbH, Lahnau, Germany) was added to the suspension at a rateof 2 ml l⁻¹ to improve the spray layer on the leaf surface. Biweeklyapplications of H39 were carried out at July 22, July 24, July 28, July31, August 5, August 7, August 11, and August 18. (3) The thirdtreatment consisted of weekly application of sulphur (Thiovit-Jet,Syngenta Crop Protection B. V., Roosendaal, The Netherlands; a.i. 80%sulphur) at a rate of 0.4% (400 gram per 100 liter water at 1000 lha⁻¹). Sulphur treatments were applied at July 22, July 28, August 5,and August 11.

The number of scabbed leaves and the number of scab spots per leaf wereassessed for the leaves of 10 shoots of each of the 4 trees per plotbefore the experiment started at June 11. In total 227 to 239 leaveswere examined per plot. At September 19, scab symptoms were assessed on177 to 232 leaves per plot which had been produced after the beginningof the experiment. Disease severity (% leaf surface covered with scabsymptoms) was estimated using following classes: 1: No scab; 2: 1-10%coverage; 3: 11-50% coverage; and 4: 51-100% coverage. A severity indexwas calculated using the formula:DS=(0×N ₁+1×N ₂+2×N ₃+3×N ₄)/N _(total)*100

In which N₁, N₂, N₃, and N₄ is the number of leaves grouped in theclasses 1, 2, 3, and 4, respectively, and N_(total) is the total numberof leaves assessed per plot.

Conidia production of V. inaequalis was assessed on susceptible youngleaves developed during the course of the experiment at 3 samplingdates. Sampling dates were chosen so that sets of susceptible leavespresent during a predicted infection period were sampled approximately 5weeks after the infection period. The Mills table based on leaf wetnessduration and temperature was used to predict infection periods. Thesecond youngest just unfolded leaf was labelled 1 to 3 days after apredicted infection period on a set of 3 twigs belonging to the sametree in each plot. The period between predicted infection infection andfirst application of C. cladosporioides H39 as well as the number ofsprays and period of protection by sprays differed per sampling date(FIG. 3). After 35 days, the 2 leaves just unfolded at the date oflabelling and the next 2 younger leaves, unfolded after labelling butexpanded during the course of the experiment, were sampled resulting ina sample consisting of 12 leaves per plot. Sampling dates were August 22(of leaves labelled on July 18), August 26 (of leaves labelled on July22), and September 4 (of leaves labelled on August 1). The 12 leaves persampled were pooled and put into 250-ml glass bottles. Within 2 h,100-0.150 ml (depending on leaf mass) of tap water with 0.01% Tween 80was added and bottles were shaken with a flask shaker at 700 OCS min⁻¹for 10 min. From the obtained suspensions, sub-samples of 6 ml werestored at −18° C. The concentration of conidia of V. inaequalis wasdetermined for each suspension with the aid of a haemocytometer. Theleaf surface of all leaves per sample was measured with an area meter.

Results and Discussion

Conidia of C. cladosporioides H39 produced in spring 2008, formulated asa water dispersible granule following the pilot protocol and stored at5° C. had a viability of 65% at the beginning of the experiments.Viability remained stable until the end of the experiment.

Before the experiment started, scab incidence on leaves did not differfor plots belonging to the different treatments. The mean incidence was81.7% in plots used as control, 84.6% for plots later treated with H39,and 84.3% for plots later treated with sulphur. Also the number of leafspots per leaf did not differ statistically, so that it can be assumedthat the scab development was similar in the different plots before theexperiment started.

On leaves sampled on August 22, August 26 and September 4 from untreatedtrees 35,242 (11,447 to 74,870), 30,242 (18,926 to 59,497) and 32,533(19,698 to 46,350) conidia of V. inaequalis·cm⁻² leaf surface wereproduced on average (backtransformed means, range for 6 replicates inbrackets) (FIG. 4). On leaves of trees treated with C. cladosporioidesH39, the number of spores was statistically significantly lower with11,499 conidia of V. inaequalis·cm⁻² leaf surface (67% reduction basedon backtransformed values) on the first sampling date and 15,139conidia·cm⁻² leaf surface (50% reduction) on the second sampling date.For the last sampling date, no significant effect was observed forapplications of C. cladosporioides H39 with 21,163 conidia·cm⁻² leafsurface (35% reduction) on treated leaves. Applications of sulphurresulted in a reduction of the number of conidia of V. inaequalisproduced per cm² leaf surface by 2, 16, and 26% for the differentsampling dates (FIG. 4). Scab severity, assessed at September 19, onemonth after the last treatment with H39, was 2.2 for the controltreatment, but statistically significantly lower with 1.8 forH39-treated plots (FIG. 5). In sulphur treated plots, scab severity was2.0 which did not differ significantly from the other treatments.

During the orchard experiment environmental conditions resulted in ahigh number of infection periods and scab development was favoured. Alsothe exceptional development of new shoots after the hail event supportedthe summer epidemic. Under such a severe disease pressure treatmentswith sulphur often are not sufficient to achieve disease control as alsofound in our experiment. Treatments with the antagonist H39 reducedconidia production of V. inaequalis under such severe conditions,confirming results from orchard experiments carried out in 2006 and2007. The strongest effect was found for the first sampling date whentreatments with H39 started after the predicted infection but werecontinued until 4 days before sampling. For the third sampling date,multiple treatments with H39 had been carried out already before thepredicted infection period but the last treatment had been applied 17days before sampling. In this situation, conidia production of V.inaequalis was reduced only by 35%. Since the effect of the antagonistmay also depend on environmental factors which differed before thedifferent sampling dates, more data are needed from repeated orchardexperiments before conclusions on optimum timing of antagonistapplications can be drawn.

For the first time also scab severity has been assessed after treatmentswith the antagonist H39. The reduced scab severity at the high scablevel observed in the orchard demonstrated that C. cladosporioides H39has a high potential to control scab epidemics.

Example 5 Orchard Assay in 2007/2008: Effect of Treatments withCladosporium cladosporioides H39 and 8406 During Summer on AscosporeProduction of Venturia inaequalis on Treated Leaves after Overwintering

Objective

Treatments of apple leaves during summer with conidial suspensions ofthe antagonists Cladosporium cladosporioides H39 and R406 led to thereduction of conidia production of the scab fungus Venturia inaequalison treated apple leaves under orchard conditions (see Example 1 and 2).

Objective of an additional assessment made on leaves treated in theorchard during the experiment in 2007 was to study the effect of suchtreatments of young developing leaves with the antagonists during summeron the ascospore production by V. inaequalis in treated leaves afteroverwintering.

Experimental design. The experiment was carried out in the organicallymanaged orchard at Applied Plant Research, Randwijk, The Netherlands.The aim of the experiment was to assess the potential of antagonistapplications to control the summer epidemic of apple scab and to reducethe inoculum load of V. inaequalis produced in overwintering appleleaves. Therefore it was essential to allow an initiation of a mild tomoderate epidemic in the orchard during the primary season. Unusualweather conditions during the primary season of 2007 with a period of 4weeks without any rain were very unfavourable for V. inaequalisascospore release and infection. Weather became more conducive afterApril and a mild apple scab epidemic had developed when the firstantagonist suspension was applied. No fungicide treatments were carriedout to reduce the progression of the epidemic before or during theexperiment.

The experiment was carried out on 8 year-old trees cv Jonagold in adesign with 6 blocks, with 2 blocks in the same tree row. Each blockconsisted of 3 plots, each with 4 trees. Between plots, 2 untreatedtrees served as buffer. The 3 different treatments were randomlyallocated to the plots. Trees were treated at a rate of 21 per plottwice per week with the following treatments: (1) tap water amended withTween 80 (0.01%) as control; and (2) conidial suspension of formulatedH39 (2×10⁶ conidia ml⁻¹). Applications were carried out twice per weekat 16 dates between 28 June and 20 August. (3) The third treatmentconsisted of multiple spray applications of conidial suspensions of C.cladosporioides R406. Conidia freshly produced at each application datewere applied twice per week on the 4 trees of each replicate plot at 12dates between 12 July and 20 August.

Conidia production of V. inaequalis during summer. The effect oftreatments on the conidia production of V. inaequalis has been reportedas Example 2.

Ascospore production of V. inaequalis in overwintering apple leaves. On12 Jul. 2007 the second youngest just unfolded leaf was labelled on 25twigs per plot. In some plots fewer twigs were available. On 4 Oct.2007, leaves were sampled which had been just unfolded at the date oflabelling and the next 2 younger leaves, unfolded after labelling.Depending on availability of leaves at the sampling date, 16 to 65leaves per plot were collected. Sampled leaves of each plot wereweighted and fixed between 2 iron nettings sized 50×50 cm (15×20 mmmesh) so that leaves did not touch each other. Nettings with leaves wereplaced on the orchard floor on bare soil within apple rows of theorganic orchard at Randwijk and fixed with metal nails so that most ofthe netting surface touched the soil. There were 6 blocks (replicates)and nettings containing leaves from different treatments (applied duringsummer) were randomly distributed within blocks.

On Feb. 26, 2008, nettings were collected. Leaf residues of bothnettings per replicate were pooled, air-dried (20° C., 70% RH, 2 days)and weighted. Subsequently, the air-dried leaf residues of each samplewere spread in separate plastic trays (50×30×6 cm) on moist filterpaper. Trays were covered by a plastic bag and leaf residues wereincubated in these moist chambers for 7 days at 20° C. in the darkfollowed by 14 days at 20° C. with 12 h light (75 □E) per day to allowmaturation (of a substantial fraction) of asci. After incubation, leafresidues were transferred into 1000 ml-plastic bottles containing150-350 ml water depending on the amount of leaf residues. Air (250 lh⁻¹) was bubbled through the water resulting in heavy turbulence during2 h (Heye et al. 1981, Canadian Journal of Botany 59, 965-968; Philion,1995, Msc Thesis McGill University, Montreal, Canada). Thereafter, theresulting suspension was passed through a sieve (1 mm mesh) to removeleaf debris. Two sub-samples (8 ml) of the suspension were stored at−20° C. Ascospore concentration in the suspensions was determinedmicroscopically using a haemocytometer. Ascospore production wasexpressed as production per g leaves (originally fixed on the orchardfloor in autumn) or per g air-dried leaf residue present at samplingdate in spring.

Results and Discussion

On leaf residues from the control treatment, 220,089 ascospores wereproduced per g leaf residue (dry weight). This was equivalent to 37,171ascospores produced per g leaf tissue (fresh weight) present in thepreceding autumn (Table 9). In residues from leaves which had beentreated with C. cladosporioides H39 in the preceding summer, only109,044 ascospores were produced, equivalent to 15,733 ascosporesproduced per g leaf tissue (fresh weight) present in the precedingautumn. This reduction of ascospore production by 58% respectively 50%compared to the control treatment was statistically significant. Theeffect of C. cladosporioides R406 on ascospore production in springafter treatment of apple leaves during the preceding summer was lesspronounced and not statistically significant.

It can be concluded that summer treatments of orchards during summerwith the antagonist C. cladosporioides H39 and possibly also with C.cladosporioides R406 have a long-lasting effect in the following springon the production of ascospores by V. inaequalis. A reduced number ofascospores produced on overwintering leaves will result in a lowerinoculum pressure during the primary infection season of apple scab andthus a slower development of scab at the beginning of the epidemic. Toour knowledge this is the first report demonstrating that treatments ofyoung developing apple leaves, highly susceptible to scab, with anantagonist results in a significantly lower ascospore production on thetreated leaves in the next season after overwintering.

Example 6 Effect of Cladosporium cladosporioides H39 on Spore Productionof Various Pathogens

Objective

The objective of subsequent experiments was to evaluate the effect ofthe antagonist on various other plant pathogens.

Materials and Methods

Conidia of Cladosporium cladosporioides H39. Conidia of Cladosporiumcladosporioides H39 were produced in a Solid-State Fermentation (SSF)system by PROPHYTA Biologischer Pflanzenschutz GmbH, Germany. Theharvested conidia were formulated as a water dispersible granule (WG).For preservation of the product quality, the final product was stored at−20° C. Viability of dried conidia was determined on malt extract agar(1 g malt extract l⁻¹) before the beginning of the experiments. Conidiaincubated for 24 hours at 20° C. with germ tubes longer than half of theminimum diameter of a conidium were considered to be viable. Sixty fivepercent of the conidia were viable.

Pathogens. The following pathogen isolates were used in the study (withgrowth medium used for spore production in brackets): Nectria galligena780 (oat meal agar; OA) causing canker in fruit trees and fruit rot,Stemphylium versicarium 850 (oat meal agar, OA) causing Brown SpotDisease and fruit rot in pear, Botrytis aclada 008 (OA) causing onionneck rot, Botrytis cinerea 143 (OA) causing grey mould in various crops,Mycosphaerella fijiensis 78 (potato dextrose agar; PDA), causing BlackSigatoka in banana, Fusarium graminearum 820 (PDA) causing Fusarium HeadBlight in cereals, Fusarium culmorum 807 (PDA) causing Fusarium HeadBlight in cereals, and Alternaria brassicicola 177 (PDA) causingdiseases in Brassiceae. All fungi were incubated for 14 days at 24° C.with 12 hrs blacklight per day. To obtain spore suspensions, cultureswere flooded with sterile tap water containing 0.01% Tween 80. Aftergently rubbing with a rubber spatula to remove spores from fungalcultures, suspensions were filtered through sterile nylon gauze with amesh of 200 □m. Concentrations of spore suspensions were determined withthe aid of a haemocytometer and adjusted with sterile tap watercontaining 0.01% (v/v) Tween 80 to 1×10² spores ml⁻¹ and 1×10³ sporesml⁻¹. For experiments with Mycosphaerella fijiensis also mycelialfragments, obtained after sterile grinding of the mycelial mass, wereincluded in the suspensions. Only mycelial fragments with more thanthree cells were considered when the concentration of the suspensionswas adjusted.

Experimental design. Separate experiments were carried out for eachpathogen and substrate type and each experiment was repeated.

Symptomless green leaves of pear, onion, rose, cyclamen, Pelargonium,banana and white cabbage were removed from greenhouse- or field-grownplants, and detached leaves were dried for several days at roomtemperature. Dry leaves were cut into segments each sized approximately2×2 cm long, sealed in plastic bags and sterilised by gamma radiation of40 kiloGray. Four-cm long segments of wheat straw (with 1 node) and ofapple twigs (with 1 branch) were processed in the same way.

Segments of leaves, straw or twigs for use in bioassays were washed withsterile tap water to remove soluble nutrients. Therefore, approximately80 segments of each substrate type were put in 150-ml aliquots ofsterile tap water contained in sterile 250 ml-conical flasks over nightat 4° C. Thereafter, segments were blotted dry with sterile filterpaper. Four segments were placed in each of a series of moist chambers.Each chamber consisted of a sterile plastic petri dish (90 mm diameter)containing two sterile filter papers (85 mm diameter) and 6 ml steriletap water. In each experiment five replications (petri dishes) fortreatments with the antagonist Cladosporium cladosporioides H39 and fivereplications for the water control were applied at each of the twolevels of pathogen application (1×10² spores ml⁻¹ and 1×10³ sporesml⁻¹). During the experiment, petri dishes were arranged in a completelyrandomised design.

Spore suspensions (or mycelial fragments in one case) of pathogens weresprayed on the segments of leaves, straw or twigs in the moist chambers.Nectria galligena was applied on apple twig segments, Stemphyliumvesicarium on leaf segments of pear, Botrytis aclada on onion leafsegments, Botrytis cinerea on leaf segments of roses, cyclamen andPelargonium, Mycospharella fijiensis on leaf segments of banana, F.graminearum and F. culmorum on straw segments, and Alternariabrassicicola on leaf segments of white cabbage. Treated tissue segmentswere incubated at 24° C. for 18 hrs. Immediately after the firstincubation period, spore suspensions of Cladosporium cladosporioides H39(2×10⁶ vital spores ml⁻¹), or water, were sprayed on the segments. Thepathogens and antagonists were applied with sterile atomisers atapproximately 5 □l cm⁻² leaf segment. Thereafter, leaves were furtherincubated at 24° C. in the dark in the moist chambers. The strawsegments with F. graminearum and F. culmorum were incubated from day 8to day 13 with 12 hrs per day blacklight. Twigs with Nectria galligenawere incubated from day 12 to day 14 with 12 hrs per day blacklightAfter a total incubation period of 9 days for Botrytis aclada andAlternaria brassicicola and 12 days for Botrytis cinerea (counted fromthe day on which pathogens were applied), the surface area of leafsegments covered with conidiophores of Botrytis aclada, Botrytis cinereaor Alternaria brassicicola was estimated using classes from zero tofive, that represented, respectively, 0%,1-5%, >5-25%, >25-50%, >50-75%, and >75-100% of the leaf surface coveredwith spore producing structures of the pathogens (Köhl et al. 1995,European Journal of Plant Pathology 101, 627-637). In the case ofStemphylium vesicarium, no conidiophores were produced, but the perfectstage (Pleospora allii) produced abundant pseudothecia. The coverage ofthe leaf surface with pseudothecia was estimated 16 days afterinoculation using the same classes as for the coverage withconidiophores. From the number of leaf segments of each class (n₀₋₅) asporulation index (SI) ranging from zero to hundred was calculated foreach replication (petri dish) consisting of four leaf segments(SI=(0×n₀+5×n₁+25×n₂+50×n₃+75×n₄+100×n₅)/4). The number of coloniesproduced by Mycosphaerella fijiensis on banana leaf segments was countedafter an incubation period of 15 days. The number of conidia of F.graminearum and F. culmorum produced on straw segments after 13 days wascounted using a microscope. Therefore, straw segments from each petridish were placed in an Erlenmeyer flask (100 ml) containing 10 ml of awashing liquid (20% ethanol in tap water containing 0.01% Tween 80).Flasks were shaken on a reciprocal shaker for 10 min, and theconcentration of conidia in the suspensions was determinedmicroscopically for F. graminearum and F. culmorum using ahaemocytometer. The same method was used to quantify the number ofconidia of Nectria galligena on apple twig segments after an incubationperiod of 14 days.

Statistics. Data were analysed separately for each experiment by ANOVAfollowed by LSD-tests. If antagonist treatment x pathogen concentrationinteractions were not significant, the mean antagonist treatment effectis analysed as main effect. Data of spore counts were log-transformedbefore analysis: log-number of spores=log₁₀(number of spores+0.01).

Results and Discussion

Nectria galligena. A strong antagonistic effect of Cladosporiumcladosporioides H39 was found against Nectria galligena causing cankerin fruit trees and fruit rot. In orchards, the pathogen survives andmultiplies on infected wound areas (cankers) of twigs (McCracken et al.,2003, Plant Pathology 52, 553-566). The inoculum produced in thebioassays on such tissues under controlled conditions was reduced by theantagonist by more than 99% (Table 10).

The results show that Cladosporium cladosporioides H39 has a highpotential for use in biocontrol of fruit tree canker which is besidesscab the most important disease in apple and pear production.Applications of the antagonist have thus the potential to control bothmajor diseases, scab as well as fruit tree canker, in the major pomefruit crops.

Stemphylium vesicarium. Stemphylium vesicarium is causing brown spot ofpear in major pear production areas in Europe (Llorente & Montesinos,2006). The major source of primary inoculum of the disease areoverwintering necrotic pear leaves on which pseudothecia of the perfectstage (Pleospora allii) are produced.

Applications of Cladosporium cladosporioides H39 on necrotic leaves,pre-colonised by the pathogen, significantly reduced the formation ofpseudothecia (estimated by assessment of leaf surface coverage by thefruiting bodies) in both experiments (Table 11). Biological control ofbrown spot by Cladosporium cladosporioides H39 may thus be achieved byapplications of the antagonist to the canopy or on the orchard floorafter leaf fall.

Stemphylium vesicarium was applied at a concentration of 1×10² sporesml⁻¹ and 1×10³ spores ml⁻¹ 18 hrs before H39 was applied with 2×10⁶spores ml⁻¹.

¹ Mean of 5 replicates, each with 4 leaf segments; leaf surface coveragewith pseudothecia was estimated.

² Significantly different from control treatment (LSD-test; □=0.05).

Botrytis spp. Presence of necrotic host tissues is a pre-requisite forepidemics of grey mould and other diseases caused by Botrytis spp. Ithas been demonstrated that competitive exclusion of Botrytis spp. fromnecrotic host tissues can be exploited for biocontrol of Botrytisincited diseases (Köhl et al., 2003, BioControl 48, 349-359).

Cladosporium cladosporioides H39 significantly reduced sporulation ofBotrytis aclada and Botrytis cinerea in bio-assays on tissues of all ofthe four different hosts tested (Tables 12-15). The antagonist has thusa potential for biological control of Botrytis-incited diseases invarious crops.

Mycosphaerella fijiensis. Mycosphaerella fijiensis is causing BlackSigatoka in banana (Arzanlou et al., 2007, Phytopathology 97,1112-1118). The disease is the major threat in banana production.Disease control at present depends on multiple fungicide applications.The pathogen survives and multiplies in necrotic lesions on leaves.

The results of the bio-assay on necrotic banana leaves show that theantagonist Cladosporium cladosporioides H39 has a promising potentialfor biological control of the disease. Although spores were not formedby the pathogen under the experimental conditions, it clearly could bedemonstrated that the antagonist is able to exclude the pathogen fromsubstrate colonisation (Table 16).

Fusarium graminearum and Fusarium culmorum. Fusarium graminearum andFusarium culmorum are causing Fusarium Head Blight in cereals and maize.The disease is causing yield losses and, often more important, severequality losses due to formation of mycotoxins by the pathogens. Strawstubbles and other debris are the main inoculum source of the disease(Osborne & Stein, 2007, International Journal of Food Microbiology 119,103-108).

Cladosporium cladosporioides H39 significantly reduced conidiaproduction of both Fusarium spp. on straw segments pre-colonised by thepathogens (Table 17 and 18) and thus has the potential for use inbiocontrol of these major pathogens in cereal production.

Alternaria brassicicola. Black leaf spot caused by Alternariabrassicicola is a major disease of Brassiceae in vegetable production.The pathogen sporulates within infected necrotic lesions and cropsdebris (Humpherson-Jones, 1989, Annals of Applied Biology 115, 45-50).Besides leaves, also whole seedlings as well as pods includingdeveloping seeds in seed production fields can be damaged.

A strong significant reduction of the conidia production of Alternariabrassicicola was found in the bio-assays after application ofCladosporium cladosporioides H39 to pre-colonised necrotic leaves ofwhite cabbage (Table 19).

Example 7 Effect of Cladosporium cladosporioides H39 on Fruit Rot ofApple

Objective

The objective of a subsequent experiment was to evaluate the effect ofthe antagonist on Monilia fructigena causing post-harvest fruit rot onapple.

Material and Methods

Conidia of Cladosporium cladosporioides H39. Conidia of Cladosporiumcladosporioides H39 were produced as in Example 6.

Pathogen. Monilia fructigena 1067, causing fruit rot in various fruitcrops, was grown on oat meal agar for 14 days at 24° C. with 12 hrsblacklight per day. To obtain spore suspensions, cultures were floodedwith sterile tap water containing 0.01% Tween 80. After gently rubbingwith a rubber spatula to remove spores from fungal cultures, suspensionswere filtered through sterile nylon gauze with a mesh of 200 □m. Sincesporulation was poor, also mycelial fragments, obtained after sterilegrinding of the mycelial mass, were included in the suspensions. Onlymycelial fragments with more than three cells were considered when theconcentration of the suspensions was determined with the aid of ahaemocytometer and adjusted with sterile tap water containing 0.01%(v/v) Tween 80 to 1×10² spores and mycelial fragments ml⁻¹ and 1×10³spores and mycelial fragments ml⁻¹.

Experimental design. Symptomless organically produced apples cv. Elstarwere used in the bio-assays. Apples were surface-sterilised bysubmerging in 70% ethanol for 1 min and rinsed three-times in steriletap-water. Four apples were placed into moist chambers (sized 28×15×9cm; with moistened filter paper on bottom; enclosed by a lid) and twowounds (approximately 3 mm in diameter and 5 mm deep) were made usingsterile cocktail pickers (Jijakli & Lepoivre, 1992, In: Fokkema, N. J.,J. Kohl & Y. Elad, Biological control of foliar and post-harvestdiseases, IOBC/WPRS Bulletin Vol. 16(11), pp. 106-110.). Fifty □l ofsterile water were applied to wounds of the control treatments. Inanother treatment, 50 □l of a conidial suspension of Cladosporiumcladosporioides H39 (2×10⁶ vital spores ml⁻¹) were added to each wound.Treated apples were incubated at 24° C. for 24 hrs. Subsequently, appleswere treated with suspensions of spores and mycelial fragments ofMonilia fructigena at 1×10² and 1×10³ spores and mycelial fragments andwere further incubated in the moist chambers at 24° C. Each treatmentwas carried out in 5 replicates, each replicate consisting of 4 applesin the same moist chamber, and moist chambers with various treatmentswere arranged in a completely randomised design. The experiment wasrepeated twice. The diameter of lesions developed from the inoculatedwounds was measured 8 and 12 days after application of Moniliafructigena.

Results and Discussion

Monilia fructigena caused severe fruit rot on wounds of apple fruitsunder the experimental conditions, e.g. lesions were produced in thecontrol treatment of both experiments on all wounds at the high pathogenlevel. Application of conidia of Cladosporium cladosporioides H39reduced the number of infected wounds in most cases (Table 20a). Theaverage lesion size was reduced by antagonist treatments by up to 70%(Table 20b). On average, fruit rot (measured as lesion size) wasstatistically significantly reduced by 50% in the first experiment, andby 30% in the second experiment.

The results demonstrate that applications of Cladosporiumcladosporioides H39 during the growing season have the potential toprotect apple fruits from pre-harvest and post-harvest fruit rot causedby Monilia fructigena. Also post-harvest treatments with Cladosporiumcladosporioides H39 are promising to protect fruit from post-harvest rotduring storage.

TABLE 1 Effect of Cladosporium cladosporioides H39 on conidia productionof Venturia inaequalis on apple seedlings under controlled conditions.Number of conidia cm^(−2 a) Experiment Treatment 1 (n = 8) ^(b) 3 (n =9) 9 (n = 10) 10 (n = 10) 11 (n = 10) 13 (n = 10) 14 (n = 11) Number oftreatments   8   9  10  10  10  10  11 Youngest leaves Control 1339 49151960 2393 4188 728 1480 C. cladosporioides H39 — — — —  863* (79) 330(55)  572 (61) Elder leaves Control — —  331  359 1313 144  614 C.cladosporioides H39 — — — —  354* (73) 116 (19)  162* (74) In total, 63fungal isolates were tested in 14 experiments. Only results of thepromising isolate are presented. ^(a) Backtransformed values; efficacyrelative to control treatment in brackets. ^(b) Total number oftreatments. ^(c) Statistically different from the control treatment.^(d) Not tested. In experiments 1 and 3, the 4 youngest leaves perseedling were pooled; in the other experiments, the youngest leaf wassampled separately from the 3 next eldest leaves.

TABLE 2A Effect of applications of Phoma pinodella H3, Coniothyriumcereale H33, Cladosporium sp. H35 and Cladosporium cladosporioides H39on conidia production of Venturia inaequalis under orchard conditions.Experiments 1, 5, and 6. Ln-Number of conidia cm⁻² Experiment Treatment1 5 6 Mean Youngest leaves Control 9.5 (12.8) 10.1 (24.9)  9.8 (18.0) 9.8 (18.0) C. cladosporioides H39 9.1 (8.6) 10.7 (46.1) 10.7 (42.5)10.2 (26.9) C. cladosporioides H39 formulated, low — 10.4 (32.4) 10.4(31.8) 10.4 (32.9) viability Elder leaves Control 9.2 (9.5)  9.2 (9.6) 9.3 (10.4)  9.2 (9.9) C. cladosporioides H39 9.5 (12.9) 10.2 (26.1) 9.7 (16.1)  9.8 (18.0) C. cladosporioides H39 formulated, low —  9.3(11.0)  9.7 (16.1)  9.5 (13.3) viability ^(a) Backtransformed values ×1000 in brackets.

TABLE 2 B Effect of applications of Phoma pinodella H3, Coniothyriumcereale H33, Cladosporium sp. H35 and Cladosporium cladosporioides H39on conidia production of Venturia inaequalis under orchard conditions.Experiments 2, 3, 4, 7, and 8. Ln- Number of conidia cm^(−2 a)Experiment Treatment 2 3 4 7 8 Mean Youngest leaves Control  8.2 (3.6)7.6 (2.1) 10.7 (46.3) 11.8 (132.1) 12.3 (227.3) 10.1 (25.3) C.cladosporioides H39  8.9 (6.9) 7.9 (2.7) 10.2 (25.8) 12.4 (253.8) 12.1(183.8) 10.3 (29.5) C. cladosporioides H39  7.4 (1.6) 6.9 (0.9) 10.4(31.7) 11.4 (90.4) 11.9 (147.2)  9.6* (14.6) formulated Elder leavesControl 10.6 (40.3) 9.2 (12.3)  9.2 (9.8) 11.2 (72.8) 11.8 (132.1) 10.4(33.0) C. cladosporioides H39 10.5 (35.3) 8.9 (7.5)  8.7 (5.8) 11.4(93.4) 11.9 (145.7) 10.3 (27.6) C. cladosporioides H39 10.0 (21.3) 8.6(5.5)  8.8 (6.6) 10.9 (52.3) 11.4 (90.7)  9.9* (20.5) formulated ^(a)Backtransformed values × 1000 in brackets. ^(b) Statistically differentfrom the control treatment

TABLE 3 Application dates and spore germination of applied inocula ofH39 and R406 (as estimated from counting from 1 agar plate sprayed inthe orchard). Spore germination (%) Date of application H39 R406 28 Jun.76 —  2 Jul. 26 —  5 Jul. 47 —  9 Jul. — — 12 Jul. 28 96 16 Jul. 32 9719 Jul. 28 95 23 Jul. 19 97 26 Jul. 26 94 30 Jul. 23 96  2 Aug. 16 97  6Aug. 11 90  9 Aug. 4 96 13 Aug. 4 95 16 Aug. 10 95 20 Aug. 4 73

TABLE 4 Epiphytical colonisation of apple leaves sampled at 4 Oct. 2007.Number of CFU cm⁻² leaf surface (backtransformed) Hyphal fungi differentfrom Treatment Cladosporium spp. Cladosporium spp. Yeasts Control 10051285 397 a ¹ H39 2223 1042 152 b R406  502  400 100 b (F_(prob) = 0.076)(F_(prob) = 0.069) Leaves had been treated twice per week from 28 Jun.until 20 Aug. with H39 and V301.61 (16 applications in total) or from 12Jul. until 20 Aug. with R406 (12 applications in total). ¹ Values of thesame column with common letters do not differ statisticallysignificantly (LSD; □ = 0.05).

TABLE 5 Endophytical colonisation of apple leaves sampled at 4 Oct.2007. Number of CFU cm⁻² leaf surface (backtransformed) Hyphal fungiCladosporium different from Treatment spp. Cladosporium spp. YeastsControl 1.19 b ¹ 4.97 1.95 H39 1.66 a 2.72 3.75 R406 1.20 b 2.93 1.99(F_(prob) = 0.182) (F_(prob) = 0.171) Leaves had been treated twice perweek from 28 Jun. until 20 Aug. with H39 and V301.61 (16 applications intotal) or from 12 Jul. until 20 Aug. with R406 (12 applications intotal). ¹ Values of the same column with common letters do not differstatistically significantly (LSD; □ = 0.05).

TABLE 6 Effect of spray applications of antagonist R406 on apple leavesin autumn 2005 on content of DNA of V. inaequalis in spring asdetermined by species-specific real-time PCR (TaqMan-PCR). Leaf residueswere sampled on 23 to 31 Jan. 2006. pg DNA of V. inaequalis per mg dryweight of leaf Treatment residues in residues of 80 leaves Control 37210093 R406 226  5007 * ¹ ¹ Statistically significantly different fromthe control treatment (LSD_(5%) = 5057).

TABLE 7 Effect of spray applications of antagonist R406 on apple leavesin autumn 2005 on V. inaequalis ascospore production on leaf residues of80 leaves in spring 2006. Number of ascospores per 80 leaves Log₁₀-Back- Relative to Treatment transformed transformed control ¹ Control5.88 749894 100 R406 5.37 * ² 235505  31 ¹ At back-transformed scale. ²Statistically significantly different from the control treatment(LSD_(5%) = 0.3604).

TABLE 8 Effect of spray applications of antagonist R406 on apple leavesin autumn 2005 on V. inaequalis ascospore production per gram of leafresidues in spring 2006. Number of ascospores per g leaf residue Log₁₀-Back- Relative to Treatment transformed transformed control ¹ Control4.54 34316 100 R406 4.07 * ² 11722  34 ¹ At back-transformed scale. ²Statistically significantly different from the control treatment(LSD_(5%) = 0.3183).

TABLE 9 Effect of treatments of young leaves with Cladosporiumcladosporioides H39 and R406 carried out during summer between Jun. 28and Aug. 20 on ascospore production of Venturia inaequalis in treatedleaves after overwintering on the orchard floor. Orchard experiment2007/2008. Log-Number of ascospores g⁻¹ of original leaf sample leafresidues in spring Treatment (fresh weight) (dry weight) Control 4.57(37,171) ^(a) 5.34 (220,089) H39 4.20 * ^(b) (15,733) 5.04 * (109,044)R406 4.52 (32,991) 5.32 (211,203) ^(a) Backtransformed values. ^(b)Significantly different from control treatment (one-sided LSD-test; a =0.05).

TABLE 10 Effect of Cladosporium cladosporioides H39 on sporulation ofNectria galligena on apple twig segments incubated in moist chamber at24° C. for 14 days. Log-number of conidia per straw segment ¹ Treatment1 × 10² spores ml⁻¹ 1 × 10³ spores ml⁻¹ Mean Experiment 1 Water control6.64 (4,365,200) 6.56 (3,630,800) 6.60 (3,981,100) H39 3.76    (5,800)3.06    (1,100) 3.41 * ²    (2,600) Experiment 2 Water control 6.59(3,881,500) 6.75   (562,300) 6.67 (4,666,600) H39 4.32   (20,900) 4.44  (27,200) 4.38 *   (23,800) Nectria galligena was applied at aconcentration of 1 · 10² spores ml⁻¹ and 1 · 10³ spores ml⁻¹ 18 hrsbefore H39 was applied with 2 × 10⁶ spores · ml⁻¹. ¹ Mean of 5replicates, each with 4 twig segments; backtransformed values inbrackets. ² Significantly different from control treatment (LSD-test; □= 0.05).

TABLE 11 Effect of Cladosporium cladosporioides H39 on sporulation ofPleospora allii (Stemphylium vesicarium) on necrotic pear leavesincubated in moist chamber at 24° C. for 16 days. Sporulation index ofPleospora allii ¹ Treatment 1 × 10² spores ml⁻¹ 1 × 10³ spores ml⁻¹ MeanExperiment 1 Water control 29.5 36.8 33.1 H39  8.2 26.8 17.5 * ²Experiment 2 Water control 49.0 48.8 48.9 H39 11.5 18.2 14.9 * ²

TABLE 12 Effect of Cladosporium cladosporioides H39 on sporulation ofBotrytis aclada on necrotic onion leaves incubated in moist chamber at24° C. for 9 days. Sporulation index of Botrytis aclada ¹ Treatment 1 ×10² spores ml⁻¹ 1 × 10³ spores ml⁻¹ Mean Experiment 1 Water control 93.898.8 96.2 H39 10.0 12.0 11.0 * ² Experiment 2 Water control 92.5 83.087.8 H39  8.8 18.0 13.4 * Botrytis aclada was applied at a concentrationof 1 × 10² spores ml⁻¹ and 1 × 10³ spores ml⁻¹ 18 hrs before H39 wasapplied with 2 × 10⁶ spores ml⁻¹. ¹ Mean of 5 replicates, each with 4leaf segments. ² Significantly different from control treatment(LSD-test; □ = 0.05).

TABLE 13 Effect of Cladosporium cladosporioides H39 on sporulation ofBotrytis cinerea on necrotic rose leaves incubated in moist chamber at24° C. for 12 days. Sporulation index of Botrytis cinerea ¹ Treatment 1× 10² spores ml⁻¹ 1 × 10³ spores ml⁻¹ Mean Experiment 1 Water control76.2 85.0 80.6 H39 17.5 43.0 30.2 * ² Experiment 2 Water control 61.596.2 78.9 H39 35.8 85.0 60.4 * Botrytis cinerea was applied at aconcentration of 1 × 10² spores ml⁻¹ and 1 × 10³ spores ml⁻¹ 18 hrsbefore H39 was applied with 2 × 10⁶ spores ml⁻¹. ¹ Mean of 5 replicates,each with 4 leaf segments. ² Significantly different from controltreatment (LSD-test; □ = 0.05).

TABLE 14 Effect of Cladosporium cladosporioides H39 on sporulation ofBotrytis cinerea on necrotic cyclamen leaves incubated in moist chamberat 24° C. for 12 days. Sporulation index of Botrytis cinerea ¹ Treatment1 × 10² spores ml⁻¹ 1 × 10³ spores ml⁻¹ Experiment 1 Water control 95.095.0 H39 28.2 * ² 66.5 * Experiment 2 Water control 95.0 90.0 H39 57.8 *95.5 Botrytis cinerea was applied at a concentration of 1 × 10² sporesml⁻¹ and 1 × 10³ spores ml⁻¹ 18 hrs before H39 was applied with 2 × 10⁶spores ml⁻¹. ¹ Mean of 5 replicates, each with 4 leaf segments. ²Significantly different from control treatment (LSD-test; □ = 0.05).

TABLE 15 Effect of Cladosporium cladosporioides H39 on sporulation ofBotrytis cinerea on necrotic Pelargonium leaves incubated in moistchamber at 24° C. for 12 days. Sporulation index of Botrytis cinerea ¹Treatment 1 × 10² spores ml⁻¹ 1 × 10³ spores ml⁻¹ Mean Experiment 1Water control 34.8 42.0 38.4 H39  1.5  8.5  5.0 * ² Experiment 2 Watercontrol 24.5 23.2 23.9 H39  0.2 11.0  5.6 * Botrytis cinerea was appliedat a concentration of 1 × 10² spores ml⁻¹ and 1 × 10³ spores ml⁻¹ 18 hrsbefore H39 was applied with 2 × 10⁶ spores ml⁻¹. ¹ Mean of 5 replicates,each with 4 leaf segments. ² Significantly different from controltreatment (LSD-test; □ = 0.05).

TABLE 16 Effect of Cladosporium cladosporioides H39 on sporulation ofMycosphaerella fijiensis on necrotic banana leaves incubated in moistchamber at 24° C. for 15 days. Number of colonies of Mycosphaerellafijiensis Treatment per leaf segment ¹ Experiment 1 Water control 5.65H39 0.15 * ² Experiment 2 Water control 10.15 H39 0.00 * Mycosphaerellafijiensis was applied at a concentration of 1 × 10³ mycelial fragmentsml⁻¹ 18 hrs before H39 was applied with 2 × 10⁶ spores ml⁻¹. ¹ Mean of 5replicates, each with 4 leaf segments. ² Significantly different fromcontrol treatment (LSD-test; □ = 0.05).

TABLE 17 Effect of Cladosporium cladosporioides H39 on sporulation ofFusarium graminearum on wheat straw incubated in moist chamber at 24° C.for 13 days. Log-number of conidia per straw segment ¹ Treatment 1 × 10²spores ml⁻¹ 1 × 10³ spores ml⁻¹ Experiment 1 Water control 5.615(412,100) 5.731 (538,300) H39 4.759 * ²  (57,400) 5.501 (317,000)Experiment 2 Water control 5.872 (744,700) 5.885 (767,400) H39 5.416 *(260,600) 5.670 * (467,700) Fusarium graminearum was applied at aconcentration of 1 × 10² spores ml⁻¹ and 1 × 10³ spores ml⁻¹ 18 hrsbefore H39 was applied with 2 × 10⁶ spores ml⁻¹. ¹ Mean of 5 replicates,each with 4 straw segments; backtransformed values in brackets. ²Significantly different from control treatment (LSD-test; □ = 0.05).

TABLE 18 Effect of Cladosporium cladosporioides H39 on sporulation ofFusarium culmorum on wheat straw incubated in moist chamber at 24° C.for 13 days. Log-number of conidia per straw segment ¹ Treatment 1 × 10²spores ml⁻¹ 1 × 10³ spores ml⁻¹ Mean Experiment 1 Water 6.496(3,133,290) 6.614 (4,111,500) 6.555 (3,589,200) control H39 6.092(1,235,900) 6.390 (2,454,700) 6.241 * ² (1,741,800) Experiment 2 Water6.446 (2,792,500) 6.521 (3,318,900) 6.484 (3,047,900) control H39 6.222(1,667,200) 6.413 (2,588,200) 6.317 * (2,074,900) Fusarium culmorum wasapplied at a concentration of 1 × 10² spores ml⁻¹ and 1 × 10³ sporesml⁻¹ 18 hrs before H39 was applied with 2 × 10⁶ spores ml⁻¹. ¹ Mean of 5replicates, each with 4 straw segments; backtransformed values inbrackets. ² Significantly different from control treatment (LSD-test; □= 0.05).

TABLE 19 Effect of Cladosporium cladosporioides H39 on sporulation ofAlternaria brassicicola on necrotic white cabbage leaves incubated inmoist chamber at 24° C. for 9 days. Sporulation index of Alternariabrassicicola ¹ Treatment 1 × 10² spores ml⁻¹ 1 × 10³ spores ml⁻¹ MeanExperiment 1 Water control 72.5 80.0 76.2 H39  2.5 23.2 12.9 * ²Experiment 2 Water control 78.8 87.5 H39 11.5 * 41.8 * Alternariabrassicicola was applied at a concentration of 1 × 10² spores ml⁻¹ and 1× 10³ spores ml⁻¹ 18 hrs before H39 was applied with 2 × 10⁶ sporesml⁻¹. ¹ Mean of 5 replicates, each with 4 leaf segments. ² Significantlydifferent from control treatment (LSD-test; □ = 0.05).

TABLE 20 Effect of Cladosporium cladosporioides H39 on fruit rot causedby Monilia fructigena on apples cv Elstar incubated in moist chamber at24° C. for 8 and 12 days. a. Number of lesions produced per 8 wounds.Number of lesions ¹ Treatment 1 × 10² spores ml⁻¹ 1 × 10³ spores ml⁻¹Mean Day 8 Water control 4.8 8.0 6.4 H39 1.8 6.6 4.2 * ² Day 12 Watercontrol 8.0 6.4 7.2 H39 7.4 5.4 6.4 b. Average lesion diameter. Lesiondiameter (mm) ¹ Treatment 1 × 10² spores ml⁻¹ 1 × 10³ spores ml⁻¹ MeanDay 8 Water control 19.0 43.1 31.1 H39  5.6 24.7 15.2 * ² Day 12 Watercontrol 53.3 85.7 69.5 H39 31.1 66.2 48.6 * Cladosporium cladosporioidesH39 was applied to wounds using 50 □l spore suspension at 2 × 10⁶ sporesml⁻¹. After 24 hrs, Monilia fructigena were applied using 50 □lsuspension at 1 × 10² spores and mycelial fragments ml⁻¹ and 1 × 10³spores and mycelial fragments ml⁻¹. ¹ Mean of 5 replicates, each with 4apples with 2 treated wounds per apple. ² Significantly different fromcontrol treatment (LSD-test; □ = 0.05).

What is claimed is:
 1. A micro-organism, Cladosporium cladosporioidesH39, as deposited on Dec. 13, 2007 under number CBS 122244 with theCentraal Bureau Schimmelcultures, Baarn, The Netherlands.
 2. Acomposition, comprising Cladosporium cladosporioides H39, as depositedon Dec. 13, 2007 under number CBS 122244 with the Centraal BureauSchimmelcultures, Baarn, The Netherlands.
 3. The composition of claim 2,wherein the Cladosporium is present as spores.
 4. The composition ofclaim 3, further comprising a carrier for the Cladosporium spores. 5.The composition of claim 4, wherein the carrier comprises glucose. 6.The composition of claim 2, wherein the Cladosporium comprises anextract of the Cladosporium.